Fusion gene, fusion protein, recombinant vector, universal DC vaccine of coronavirus and preparation method thereof

文档序号：842707 发布日期：2021-04-02 浏览：11次中文

阅读说明：本技术 冠状病毒的融合基因、融合蛋白、重组载体、通用型dc疫苗及其制备方法 (Fusion gene, fusion protein, recombinant vector, universal DC vaccine of coronavirus and preparation method thereof ) 是由焦顺昌张嵘汪坤福于 2020-12-31 设计创作，主要内容包括：本发明提供了冠状病毒的融合基因、融合蛋白、重组载体、通用型DC疫苗及其应用,属于病毒疫苗制备技术领域,融合基因包括编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD单聚体、二聚体或三聚体蛋白的基因；本发明通过在抗原提呈细胞系中表达融合基因获得一种冠状病毒通用型DC细胞疫苗；所述通用型的DC疫苗可以有效的激活T细胞；根据实施例的记载,本发明制备获得的通用型DC疫苗可以有效激活食蟹猴针对COVID-19病毒N蛋白和RBD蛋白的特异性的抗体。(The invention provides a fusion gene, a fusion protein, a recombinant vector, a universal DC vaccine and application thereof of coronavirus, belonging to the technical field of preparation of virus vaccines, wherein the fusion gene comprises a gene for coding COVID-19 virus N protein and a gene for coding COVID-19 virus RBD unimer, dimer or trimer protein; the invention obtains a coronavirus universal DC cell vaccine by expressing fusion genes in an antigen presenting cell line; the universal DC vaccine can effectively activate T cells; according to the description of the embodiment, the universal DC vaccine prepared by the invention can effectively activate the specific antibody of the cynomolgus monkey to the COVID-19 virus N protein and the RBD protein.)

1. A fusion gene of coronavirus, which comprises a gene coding a COVID-19 virus N protein and a gene coding a COVID-19 virus RBD protein; the gene coding the COVID-19 virus N protein and the gene coding the COVID-19 virus RBD protein are connected through a connecting sequence;

the gene for coding the COVID-19 virus RBD protein comprises a gene for coding an RBD protein unimer, a gene for coding an RBD protein dipolymer or a gene for coding an RBD protein tripolymer.

2. The fusion gene of claim 1, wherein the nucleotide sequence of the fusion gene is shown in any one of SEQ ID No. 1-SEQ ID No. 3.

3. The fusion protein encoded by the fusion gene of claim 1, wherein the amino acid sequence of the fusion protein is represented by any one of SEQ ID Nos. 4 to 6.

4. A recombinant vector comprising the fusion gene of claim 1 or 2 and an original expression vector.

5. The recombinant vector according to claim 4, wherein the original expression vector is a lentiviral vector.

6. Use of the fusion gene according to claim 1 or 2, the fusion protein according to claim 3, or the recombinant vector according to claim 4 or 5 for the preparation of a medicament for the prevention and/or treatment of a disease caused by a coronavirus infection.

7. A coronavirus universal DC cell vaccine obtained by expressing the fusion gene of claim 1 in an antigen-presenting cell line;

the antigen presenting cell line expresses a fusion gene of TAX and ST 40; the nucleotide sequence of the fusion gene of TAX and ST40 is shown in SEQ ID No. 7;

the antigen presenting cell line is an antigen presenting cell amplified by a limited number of generations.

8. A method of preparing the coronavirus universal DC cell vaccine of claim 7, comprising the steps of:

1) packaging a recombinant vector comprising a fusion gene of TAX and ST40 into a lentivirus to obtain a packaged virus;

2) infecting the DC cells with the packaging virus to obtain infected DC cells;

3) carrying out first co-culture on the infected DC cells and the feeder cells for 4-6 weeks, removing CD3+ cells, carrying out second co-culture for 1-2 weeks, removing CD3+ cells again, carrying out third co-culture for 2-6 months, and collecting antigen presenting cells with limited expansion generations to obtain an antigen presenting cell line;

4) transferring the recombinant vector expressing the fusion gene of coronavirus in claim 4 or 5 into the antigen presenting cell line in step 3) to obtain the universal DC cell vaccine targeting coronavirus.

9. The method according to claim 8, wherein the ratio of the number of infected DC cells to the number of feeder cells in step 3) is (0.8-1.2): (0.8 to 1.2).

10. The method of claim 8, wherein step 4) further comprises an antibiotic selection step after transferring the recombinant vector into the antigen presenting cell line.

Technical Field

The invention belongs to the technical field of preparation of virus vaccines, and particularly relates to a fusion gene, a fusion protein, a recombinant vector, a universal DC vaccine and a preparation method thereof of three coronaviruses.

Background

Coronaviruses belong to the phylogenetic group of the family of Coronaviridae (Coronaviridae) and the genus coronaviruses (Coronavirus), and human diseases caused by coronaviruses are mainly respiratory infections. The types of coronavirus vaccines in the prior art at present include inactivated coronavirus vaccines, coronavirus protuberant protein vaccines, adenovirus vector vaccines and the like.

However, no general DC cell vaccine targeting coronaviruses has been reported in the prior art.

Disclosure of Invention

In view of the above, the present invention aims to provide a coronavirus fusion gene, a coronavirus fusion protein, a recombinant vector, a universal DC vaccine, and a method for preparing the same; the fusion gene of the coronavirus comprises a gene for coding N protein and genes for coding RBD unimer, dimer and trimer protein which are respectively fused; antigen expression corresponding to N protein and RBD unimer, dimer and trimer protein can be detected in the universal DC cell prepared by the fusion gene, and the universal DC vaccine can effectively stimulate specific T cells and activate the T cells; the universal DC vaccine can effectively activate specific antibodies of the cynomolgus monkey against COVID-19 virus N protein and RBD protein.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention also provides a fusion gene of coronavirus, which comprises a gene for coding the COVID-19 virus N protein and a gene for coding the COVID-19 virus RBD protein; the gene coding the COVID-19 virus N protein and the gene coding the COVID-19 virus RBD protein are connected through a connecting sequence;

the gene for coding the COVID-19 virus RBD protein comprises a gene for coding an RBD protein unimer, a gene for coding an RBD protein dipolymer or a gene for coding an RBD protein tripolymer.

Preferably, the nucleotide sequence of the fusion gene is shown in any one of SEQ ID No. 1-SEQ ID No. 3.

Preferably, the amino acid sequence of the fusion protein is shown in any one of SEQ ID No. 4-6.

The invention provides a recombinant vector, which comprises the fusion gene and an original expression vector.

Preferably, the original expression vector is a lentiviral vector.

The invention provides application of the fusion gene, the fusion protein and the recombinant vector in preparing a medicament for preventing and/or treating diseases caused by coronavirus infection.

The invention provides a coronavirus universal DC cell vaccine, which is obtained by expressing the fusion gene in an antigen presenting cell line;

the antigen presenting cell line expresses a fusion gene of TAX and ST 40; the nucleotide sequence of the fusion gene of TAX and ST40 is shown in SEQ ID No. 7;

the antigen presenting cell line is an antigen presenting cell amplified by a limited number of generations.

The invention provides a preparation method of the coronavirus universal DC cell vaccine, which comprises the following steps:

1) packaging a recombinant vector comprising a fusion gene of TAX and ST40 into a lentivirus to obtain a packaged virus;

2) infecting the DC cells with the packaging virus to obtain infected DC cells;

4) transferring the recombinant vector of the fusion gene for expressing the coronavirus into the antigen presenting cell line in the step 3) to obtain the universal DC cell vaccine of the targeted coronavirus.

Preferably, the number ratio of the infected DC cells to the trophoblasts in the step 3) is (0.8-1.2): (0.8 to 1.2).

Preferably, the step 4) of transferring the recombinant vector into the antigen presenting cell line further comprises an antibiotic screening step.

The invention has the beneficial effects that: the fusion gene of coronavirus provided by the invention comprises a gene for coding COVID-19 virus N protein and a gene for coding COVID-19 virus RBD protein; the gene for coding the COVID-19 virus RBD protein comprises a gene for coding an RBA protein unimer, a gene for coding an RBD protein dipolymer or a gene for coding an RBD protein tripolymer; the gene coding the COVID-19 virus N protein and the gene coding the COVID-19 virus RBD unimer, dimer or trimer protein are connected through a connecting sequence. Antigen expression corresponding to N protein and RBD protein can be detected in the universal DC prepared by the fusion gene, and the universal DC vaccine can effectively stimulate specific T cells and activate the T cells; according to the description of the embodiment, the universal DC vaccine prepared by the invention can effectively activate the specific antibody of the cynomolgus monkey to the COVID-19 virus N protein and the RBD protein.

Drawings

FIG. 1 shows the result of identifying antigen expression by Western method;

FIG. 2 is a diagram showing the ratio of flow-detecting universal DC-activated T cells, in which from left to right, from top to bottom, are Control, N-RBD-monomer, N-RBD-dimer and N-RBD-trimer;

FIG. 3 shows the IgG antibodies specific to the viral antigen against the N protein in cynomolgus monkey sera.

FIG. 4 shows the viral antigen-specific IgG antibodies against RBD protein in cynomolgus monkey serum.

Detailed Description

The invention provides a fusion gene of coronavirus, which comprises a gene for coding COVID-19 virus N protein and a gene for coding COVID-19 virus RBD protein; the gene coding the COVID-19 virus N protein and the gene coding the COVID-19 virus RBD protein are connected through a connecting sequence; the gene for coding the COVID-19 virus RBD protein comprises a gene for coding an RBD protein unimer, a gene for coding an RBD protein dipolymer or a gene for coding an RBD protein tripolymer.

In the invention, the nucleotide sequence of the fusion gene is shown in any one of SEQ ID No. 1-SEQ ID No. 3. In the invention, the fusion gene shown in SEQ ID No.1 comprises a gene coding COVID-19 virus N protein and a gene coding COVID-19 virus RBD protein unimer; the fusion gene shown in SEQ ID No.2 comprises a gene for coding COVID-19 virus N protein and a gene for coding COVID-19 virus RBD protein dimer; the fusion gene shown in SEQ ID No.3 comprises a gene for coding COVID-19 virus N protein and a gene for coding COVID-19 virus RBD protein trimer; the specific sequence information is as follows:

nucleotide sequence of N-RBD-monomer (SEQ ID No. 1):

wherein the italic is linker.

Nucleotide sequence of N-RBD-dimer (SEQ ID No. 2):

where the italics are linker and the dimerization domain is underlined.

Nucleotide sequence of N-RBD-trimer (SEQ ID No. 3):

italics is linker and underlined is the trimerization domain.

The invention also provides a fusion protein coded by the fusion gene, the amino acid sequence of the fusion protein is shown as SEQ ID No. 4-6, and the specific sequence information is as follows:

N-RBD-monomer amino acid sequence (SEQ ID No. 4):

wherein the italic is linker.

N-RBD-dimer amino acid sequence (SEQ ID No. 5):

where the italics are linker and the dimerization domain is underlined.

N-RBD-trimer amino acid sequence (SEQ ID No. 6):

where the italics are linker and the underlined trimerization domain.

In the invention, the nucleotide sequence of the gene coding the COVID-19 virus N protein is preferably optimized by codon, as shown in SEQ ID No.8, the nucleotide sequence of the gene coding the COVID-19 virus RBD protein unimer is preferably optimized by codon, as shown in SEQ ID No.9, and the nucleotide sequence of the gene coding the COVID-19 virus RBD protein dimer is preferably optimized by codon, as shown in SEQ ID No.10, specifically as follows:

agggtgcagccaaccgagtctatcgtgcgctttcctaatatcacaaacctgtgcccatttggcgaggtgttcaacgcaacccgcttcgccagcgtgtacgcctggaataggaagcggatcagcaactgcgtggccgactatagcgtgctgtacaactccgcctctttcagcacctttaagtgctatggcgtgtcccccacaaagctgaatgacctgtgctttaccaacgtctacgccgattctttcgtgatcaggggcgacgaggtgcgccagatcgcccccggccagacaggcaagatcgcagactacaattataagctgccagacgatttcaccggctgcgtgatcgcctggaacagcaacaatctggattccaaagtgggcggcaactacaattatctgtaccggctgtttagaaagagcaatctgaagcccttcgagagggacatctctacagaaatctaccaggccggcagcaccccttgcaatggcgtggagggctttaactgttatttcccactccagtcctacggcttccagcccacaaacggcgtgggctatcagccttaccgcgtggtggtgctgagctttgagctgctgcacgccccagcaacagtgtgcggccccaagaagtccaccaatctggtgaagaacaagtgcgtgaacttcggcagca ccgagttcagcgaggagcagaagaaggccctggacctggccttctacttcgaccgccgcctgacccccgagtggcg ccgctacctgagccagcgcctgggcctgaacgaggagcagatcgagcgctggttccgccgcaaggagcagcagatc ggc(ii) a The dimerization domain is underlined.

In the invention, the nucleotide sequence of the gene coding the CODV-19 virus RBD protein trimer is preferably optimized by codon, as shown in SEQ ID No.11, and concretely comprises the following steps:

agggtgcagccaaccgagtctatcgtgcgctttcctaatatcacaaacctgtgcccatttggcgaggtgttcaacgcaacccgcttcgccagcgtgtacgcctggaataggaagcggatcagcaactgcgtggccgactatagcgtgctgtacaactccgcctctttcagcacctttaagtgctatggcgtgtcccccacaaagctgaatgacctgtgctttaccaacgtctacgccgattctttcgtgatcaggggcgacgaggtgcgccagatcgcccccggccagacaggcaagatcgcagactacaattataagctgccagacgatttcaccggctgcgtgatcgcctggaacagcaacaatctggattccaaagtgggcggcaactacaattatctgtaccggctgtttagaaagagcaatctgaagcccttcgagagggacatctctacagaaatctaccaggccggcagcaccccttgcaatggcgtggagggctttaactgttatttcccactccagtcctacggcttccagcccacaaacggcgtgggctatcagccttaccgcgtggtggtgctgagctttgagctgctgcacgccccagcaacagtgtgcggccccaagaagtccaccaatctggtgaagaacaagtgcgtgaacttcggttcag gcggaggttatattcctgaagctccaagagatgggcaagcttacgttcgtaaagatggcgaatgggtattgctttc taccttttta。

the invention also provides a recombinant vector which comprises the fusion gene N-RBD-RBD and an original expression vector. In the present invention, the original expression vector is a lentiviral vector, more preferably a pCDH-series vector or a plet-series vector; the fusion gene is preferably recombined between XbaI and EcoRI enzyme cutting sites of the original expression vector; the fusion gene is preferably expressed from the EF1 a promoter. The method for preparing the recombinant vector is not particularly limited in the present invention, and a conventional method for preparing a recombinant vector in the art may be used.

The invention provides application of the fusion gene, the fusion protein and the recombinant vector in preparing a medicament for preventing and/or treating diseases caused by coronavirus infection. In the present invention, the coronavirus is a COVID-19 virus; the medicament comprises a vaccine, preferably a universal DC vaccine.

The invention also provides a coronavirus universal DC cell vaccine, which is obtained by expressing the fusion gene in an antigen presenting cell line; the antigen presenting cell line expresses a fusion gene of TAX and ST 40; the nucleotide sequence of the fusion gene of TAX and ST40 is shown in SEQ ID No. 7.

In the present invention, the TAX gene and ST40 gene are preferably linked by a linker sequence; in the invention, the nucleotide sequence of the TAX gene is shown as SEQ ID NO.12, and the nucleotide sequence of the ST40 gene is shown as SEQ ID NO. 13; the nucleotide series of the fusion gene of the TAX and ST40 is shown in SEQ ID No. 7. In the present invention, the fusion gene of TAX and ST40 is used to immortalize dendritic cells and has a limited proliferation ability.

The invention also provides a preparation method of the coronavirus universal DC cell vaccine, which comprises the following steps: 1) packaging a recombinant vector comprising a fusion gene of TAX and ST40 into a lentivirus to obtain a packaged virus; 2) infecting the DC cells with the packaging virus to obtain infected DC cells; 3) carrying out first co-culture on the infected DC cells and the feeder cells for 4-6 weeks, removing CD3+ cells, carrying out second co-culture for 1-2 weeks, removing CD3+ cells again, carrying out third co-culture for 2-6 months, and collecting antigen presenting cells with limited expansion generations to obtain an antigen presenting cell line; 4) transferring the recombinant vector expressing the fusion gene into the antigen presenting cell line in the step 3) to obtain the universal DC cell vaccine of the targeted coronavirus.

In the present invention, a recombinant vector comprising a fusion gene of TAX and ST40 was packaged into a lentivirus to obtain a packaged virus. In the present invention, it is preferable that the fusion gene of TAX and ST40 is recombined into an expression vector to obtain a recombinant vector, the expression vector is preferably a plasmid (lentiviral vector) pcDH-EF1 alpha-MCS for preparing immortalized dendritic cells, and the fusion gene of TAX and ST40 is preferably recombined between EcoRI and BamHI cleavage sites of the expression vector. The method for packaging lentivirus is not particularly limited, and the conventional method in the field can be adopted.

After obtaining the packaging virus, infecting the packaging virus with a DC cell to obtain an infected DC cell; the method for infecting DC cells by the packaging virus is not particularly limited by the invention, and the conventional method in the field can be adopted.

After obtaining infected DC cells, carrying out first co-culture on the infected DC cells and trophoblasts for 4-6 weeks, removing CD3+ cells, carrying out second co-culture for 1-2 weeks, removing CD3+ cells again, carrying out third co-culture for 2-6 months, and collecting antigen presenting cells with limited expansion passage numbers to obtain an antigen presenting cell line. In the present invention, the infected DC cells are preferably co-cultured with feeder cells, which are preferably seeded in the upper culture chamber of a cell culture apparatus, preferably in an amount of 0.5X 10⁶～1.5×10⁶One/hole, more preferably 1X 10⁶Per well; the infected DC cells are preferably inoculated in the lower culture chamber of the cell culture apparatus, and the inoculation amount of the infected DC cells is preferably 0.5X 10⁶～1.5×10⁶One/hole, more preferably 1X 10⁶Per well. In the present invention, the feeder cells are preferably prepared by the following method: culturing peripheral blood mononuclear cells in a 1640 culture medium containing fetal calf serum for 9-14 h, preferably 12h to obtain trophoblasts; the mass percentage content of fetal calf serum in the 1640 culture medium is 8-12%, and the preferred mass percentage content is 10%. The antigen presenting cell can be stably amplified, and can solve the problems that the DC of a patient is difficult to obtain and culture and the genetic operation is difficult; and the limited amplification generation number is safer relative to a cell line.

After the antigen presenting cell line is obtained, the recombinant vector of the expression gene is transferred into the antigen presenting cell line to obtain the universal DC cell vaccine of the targeted coronavirus. The specific operation of the transfer is not particularly limited, and the conventional method in the field can be adopted. In the specific implementation process of the invention, the antigen presenting cell line and the recombinant vector for expressing genes (namely lentiviruses for expressing N-RBD-monomer, N-RBD-dimer and N-RBD-trimer genes) are mixed uniformly and then incubated, the incubation temperature is preferably 31-33 ℃, more preferably 32 ℃, and the incubation time is preferably 4-6 h, more preferably 5 h. The invention preferably further comprises an antibiotic screening step after the recombinant vector is transferred into the antigen presenting cell line. In the present invention, the antibiotic is preferably puromycin (2. mu.g/mL). After the incubation, the cells are collected, the cells are resuspended by using a culture solution, and the cells are mixed with puromycin and then are screened and cultured for 4-8 days to obtain the general DC cell vaccine of the targeted coronavirus. During the screening culture period, the puromycin concentration in a culture system is maintained to be 0.5-2 mu g/mL.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

See table 1 for reagent formulations in the examples below.

TABLE 1 reagent formulations

Name (R)	Formulation of
		AP	The AIMV contained 2.5% FBS by volume,
1640F	1640 contains 10% FBS, 250U/ml IL-2
		AF	The AIMV contains 10% FBS by volume, 250U/ml IL-2

Example 1

Establishment of antigen presenting cell line with limited expansion algebra

1) Synthesizing a fusion gene of TAX and ST 40;

2) connecting the fusion gene synthesized in the step 1) to an expression vector to obtain a recombinant vector;

3) packaging the lentivirus with the vector in the step 2) according to the instruction of a lentivirus packaging kit (purchased from Beijing Synbiotic Gene technology Co., Ltd.) to obtain a packaged virus, and infecting cells of the DC with the packaged virus to obtain infected cells of the DC;

4) inoculating trophoblasts into an upper culture chamber of a cell culture device, inoculating the cells of the infected DC obtained in the step 3) into a lower culture chamber of the cell culture device, culturing for 4-6 weeks, removing CD3+ cells, continuing to culture for 1-2 weeks, removing CD3+ cells, continuing to culture for more than 2 months, and obtaining the antigen presenting cell line with limited expansion passage number.

Isolation and activation of DC cells

1. Peripherical blood-derived Dendritic Cells (DCs) isolated and activated by the adherence method

1) Collecting blood singly, and separating PBMC;

2) PBMC were adjusted to 1X 10 with Medium 1640+ 10% FBS⁶cells/mL, in Petri dishes, 5% CO at 37 ℃₂An incubator stands overnight; collecting suspension cells, and marking the suspension cells as T + B for later use;

3) the cells attached to the bottom of the culture dish are blown and beaten by the culture medium 1640+ 10% FBS +100IU/mL IL-2, adherent mononuclear cells are collected, namely the DC from the peripheral blood source,

4) the phytohemagglutinin PHA of 35. mu.g/mL was added, and the cells were stimulated and cultured for 24 hours for further use.

2. Fusion gene lentivirus expression plasmid

1) Glycerol stbl3 transformed with a lentivirus expression plasmid of the fusion gene was thawed on ice, and 100. mu.l to 3ml of each LB medium containing 100. mu.g/ml of ampicillin was activated at 37 ℃ and 220rpm for 6 hours. The cells were each cultured in 100ml of LB medium containing 100. mu.g/ml ampicillin overnight at 37 ℃ and 220 rpm. Centrifuging at 4000 Xg for 10min the next day, and collecting the bacterial liquid precipitate.

2) Preparation work:

a) RNase A was added to Solution I and stored at 4 ℃.

b) To the DNA Wash Buffer was added 180ml of ethanol.

c) 76ml isopopanol (isopropanol) was added to HBC Buffer.

d) BufferN3 was placed on ice and a 42 ℃ water bath was prepared.

3) Adding 3ml GPS Buffer to the DNA chromatographic column, standing at room temperature for 4min, centrifuging at 4000 Xg for 3min, and discarding the waste liquid for later use.

4) 100ml of the bacterial sediment is taken, 10ml of Solution I/RNase is respectively added, the mixture is rotated up and down, and the mixture is sucked, beaten and mixed evenly.

5) Adding 10ml Solution II, slightly inverting for 8-10 times, and keeping the temperature at room temperature for 3 min.

6) 5ml of precooled BufferN3 was added and gently inverted 8-10 times until a white flocculent precipitate formed, which was allowed to stand at room temperature for 2 min.

7) The liquid was transferred to a new 50ml centrifuge tube using a filtration syringe and the volume was measured.

8) Adding 0.1 volume times of ETR Solution, and slightly reversing for 8-10 times.

9) The mixture was kept on ice for 10min while turning over (the liquid turned clear from turbid).

10) The water bath was carried out at 42 ℃ for 5min, and the liquid became cloudy again.

11) After centrifugation at 4000 Xg for 3min, the ETR Solution will form a layer at the bottom.

12) The supernatant was transferred to another new 50ml centrifuge tube and its volume was measured.

13) Adding 0.5 volume time of alcohol, slightly inverting for 8-10 times, and keeping the temperature at room temperature for 2 min.

14) 20ml of the above liquid was put into a DNA column, centrifuged at 4000 Xg for 3min, and the waste liquid was discarded.

15) Step 14 is repeated.

16) 10ml of HBC Buffer was added to the column, and the column was centrifuged at 4000 Xg for 3min, and then the waste liquid was discarded.

17) 15ml of DNAWash Buffer was added to the column, centrifuged at 4000 Xg for 3min, and the waste liquid was discarded.

18) 10ml of DNAWash Buffer was added to the column, centrifuged at 4000 Xg for 3min, and the waste liquid was discarded.

19) After 10min of 4000 Xg air separation, the column was placed in a new 50ml centrifuge tube and 800. mu.l dH was added dropwise to the central membrane₂O, standing at room temperature for 5min, centrifuging at 4000 Xg for 5min, and collecting the plasmid solution in a centrifuge tube.

20) Adding the plasmid solution back into the column, standing at room temperature for 5min, centrifuging at 4000 Xg for 5min, collecting the plasmid solution in a centrifuge tube, packaging, and freezing.

Sample 2. mu.l, 2. mu.l dH₂O was used as a control and the concentration was measured.

EXAMPLE 2 construction of a lentiviral vector expressing the ST40-TAX2 fusion Gene and transformation of DCs

1) Packaging the lentivirus: 293T cell plating density: 1.8X 10⁷20mL of OPTI-MEM medium was placed at 37 ℃ in 5% CO₂And (5) culturing. Amount of transfection plasmid obtained in example 1: packaging plasmid dosage: 15 μ g, P-ST40-TAX2 plasmid (preparation of immortalized Tree)Plasmid of mutant cells): 15 ug, adding the plasmid into the buffer solution, shaking and mixing for 5s, adding the transfection reagent: 60 mu L, mixing with pipettor 5 deg.C, incubating at room temperature for 10min, dropping the transfection mixture into cells, and adding 5% CO at 37 deg.C₂And (5) culturing. 3h after transfection, the medium was replaced with fresh medium, 37mL of OPTI-MEM (6% FBS) medium was added to each T175 flask, the supernatant was harvested for 96h, then the virus concentrate (purchased from Beijing Synbiotic Gene technology Co., Ltd.) was added, and after 24h of precipitation, the virus was harvested by centrifugation at 4000g for 30 min.

2) Lentivirus infection of DC: the peripheral blood-derived DCs obtained in example 1 were mixed with 1mL of 1640+ 10% FBS + 0.1% polybrene + 100. mu.L of virus supernatant in a 3X 10-fold ratio⁵Adding the cells/well into a 6-well plate, incubating at 32 ℃ for 4-6 h, and adding CO₂The culture is carried out in an incubator, and 2mL of 1640+ 10% FBS is supplemented after 4 h. Transfer to the lower layer of the well cell culture vessel by Day 3, and mark Day 0.

3) The T + B cells obtained in example 1 were resuspended in 1640+ 10% FBS +100IU/mL IL-2, and the resulting suspension was added as feeder cells to the upper culture chamber of a plug-in cell culture apparatus (12-well plate) so that the number of cells inoculated was 1X 10⁶Cells/well;

4) placing at 37 ℃ with 5% CO₂Culturing in an incubator, observing the state of cells at the lower layer every day, and changing the liquid by half when the cell liquid turns yellow;

5) counting and detecting the survival rate of the upper layer of trophoblasts every day, and replacing new trophoblasts when the survival rate of the upper layer of trophoblasts is lower than 50%;

6) cells were transferred according to growth, and the number of transferred cells was consistent with the initial number.

a) Collecting all lower-layer cells when the cells are cultured for 4-6 weeks, and removing CD3+ cells by utilizing America and whirly CD3+ sorting magnetic beads;

b) the remaining cells, 1640+ 10% FBS +100IU/mL IL-2 heavy suspension, placed at 37 degrees C5% CO₂Culturing for 1-2 weeks in an incubator;

c) detecting the phenotype of the cells by a flow cytometry analyzer, and if CD3+ cells exist, repeating the separation steps; if CD3+ cells are not present, culturing is continued in 1640+ 10% FBS +100IU/mL IL-2 medium, and after 2 months, universal DCs can be obtained.

Example 3 construction of a Universal DC vaccine targeting coronavirus N and RBD proteins

1) Entrusted biotechnology company to synthesize coronavirus N protein + RBD unimer, N protein + RBD dimer, and N protein + RBD trimer protein fusion genes;

2) connecting the synthesized fusion gene to a lentivirus expression vector to obtain a recombinant vector for expressing N-RBD-monomer, N-RBD-dimer and N-RBD-trimer genes, packaging the lentivirus, subpackaging and storing in a refrigerator at the temperature of 80 ℃ below zero;

3) transferring the recombinant vector obtained in the step 2) into the universal antigen presenting cell prepared in the example 2, and adding puromycin for screening, wherein the specific steps are as follows:

the general DC obtained in the example 2 is placed in a water bath kettle at 37 ℃ for quick thawing; transferring the mixture into a 15ml centrifuge tube, and centrifuging for 5min at 900 rpm; after centrifugation, the suspension is resuspended by 10ml and counted; taking 12-hole plate, and arranging according to 1 × 10⁶Plating culture is carried out on the wells, and culture medium supernatant is sucked and added into corresponding wells after being uniformly mixed with viruses; incubating the attacked cells for 4-6 h at the temperature of 32 ℃.

Cell culture

The first day: observing the state of the cells under a microscope; 2ml of AF liquid is supplemented to each hole of the suspension cells after the detoxification, and the suspension cells are placed at 37 ℃ and 5 percent CO₂Continuously culturing in the incubator;

and on the third day: transferring the cells of each hole into a 15ml centrifuge tube by using a pipette, and centrifuging for 5min at 1000 rpm; centrifuging, removing supernatant, re-suspending 5mL AF culture medium, transferring the re-suspended cells to T25 culture flask, adding puromycin (2 μ g/mL), mixing, adding 5% CO at 37 deg.C₂Culturing in an incubator;

the fifth day: 7mL of AF medium and puromycin (2. mu.g/mL) were added to each flask;

the sixth day: performing bottle expansion culture on the cells, transferring the cells into a 50ml centrifuge tube by using a pipette, and centrifuging for 5min at 600 rpm; centrifuging, removing supernatant, taking 20-30 ml of 1640F culture medium for resuspension, and putting the resuspended cells into a T75 culture flaskPuromycin (2. mu.g/mL) was added, shaken well and placed at 37 ℃ in 5% CO₂Culturing in an incubator;

the eighth day: the cells were cultured in expanded flasks. Transferring the cells into a 50ml centrifuge tube by a pipette, and centrifuging for 5min at 600 rpm; centrifuging, removing supernatant, re-suspending 50mL 1640F culture medium, placing the re-suspended cells into T75 culture flask, adding puromycin (2 μ g/mL), shaking, and placing at 37 deg.C and 5% CO₂Culturing in an incubator;

the tenth day: the cells were cultured in expanded flasks. Transfer the cells to a 50ml centrifuge tube with a pipette, centrifuge at 900rpm for 5 min; centrifuging, removing supernatant, resuspending 1640F culture medium, placing the resuspended cells into 1T 175(200 ml/bottle) culture flask, shaking, and placing at 37 deg.C and 5% CO₂Culturing in an incubator;

and (3) taking the cell, extracting a genome, amplifying a fusion gene sequence by using the genome as a template and using a PCR (polymerase chain reaction) method, and then carrying out sanger sequencing to obtain the universal DC cell vaccine aiming at the coronavirus fusion protein after the sequencing is correct.

Then collecting N-RBD-dimer-DC, N-RBD-trimer-DC and DC cells, centrifuging the cell sample with 4 deg.C precooled PBS twice, 2500Xg, 5min, to 2X 10⁶200. mu.l of RIPA lysate were added to each cell (the appropriate volume of Halt was added to the RIPA lysate before use)^TMProtease Inhibitor Cocktail) for 15min on ice, shaking for 30sec every 5min, pre-cooling the centrifuge at 4 ℃, centrifuging the cell lysate for 15min at 14000 Xg, transferring the supernatant to a new centrifuge tube, labeling, and storing at-80 ℃. Taking out the sample from-80 deg.C, dissolving, adding appropriate volume of 6 × Protein Loading Buffer, mixing, boiling water bath at 100 deg.C for 10min, taking out, cooling, and packaging. The expression of the antigen is detected by taking a sample and carrying out Western Blot, and the result is shown in FIG. 1: the expression amount of the N protein in the N-RBD-monomer-DC, the N-RBD-dimer-DC and the N-RBD-trimer-DC is basically consistent, and the expression amount and the presentation trend of the RBD protein are consistent with expected results.

1. T cell activation by virus antigen universal DC cell

After stimulating and culturing CTL by using the universal DC cell vaccine, adding the universal DC cell vaccineThe DC cells are subjected to secondary stimulation, the expression condition of CD137 is detected, and the result is shown in FIG. 2, wherein Control is the cells cultured without the stimulation of the general DC cells, and the result shows that: N-RBD-monomer-DC, N-RBD-dimer-DC and N-RBD-trimer-DC activated CD8⁺The proportion of T cells was: 8.05%, 13.5% and 27.9%, indicating that all 3 combinations of bivalent universal DCs were effective in activating T cells.

2. Cynomolgus monkey safety evaluation

Cynomolgus monkeys repeated subcutaneous injections of N-RBD-monomer-DC, N-RBD-dimer-DC, and N-RBD-trimer-DC, 1 administration for 2 weeks, continuous administration, and 2 total administrations. I.e., at D1 and D15. Selecting common-grade cultured cynomolgus monkeys with the initial age of about 2-5 years and the weight of about 2.5-5 kg, using 6 cynomolgus monkeys in total, and dividing the cynomolgus monkeys into 3 groups: injection dosage is 5X 10⁷cell/vehicle (highest dose corresponds to 50 times the high dose administered according to the clinical schedule) (table 2 below). The immunogenicity and the initial safety were observed.

TABLE 2 test animal groups

General clinical observation results show that after the universal DC vaccine is injected, the mental states of 1-3 groups of cynomolgus monkeys are good, and the behavioral appearance is not obviously abnormal; no obvious abnormality is found in body weight and body temperature; clinical pathological detection results show that WBC is increased to different degrees after other animals are inoculated except 3, the WBC belongs to normal immune response, and other indexes are not obviously abnormal. The biochemical detection result of the blood shows that the blood coagulation function and the biochemical indexes of the blood of 1-3 groups of animals have no obvious abnormality.

IgG antibody detection

Sera were collected prior to first dose, D7, D14, D21 and D28, respectively. Detecting changes in serum of IgG antibodies specific for the novel coronavirus N protein and RBD protein antigens.

The detection results are shown in fig. 3 and 4, fig. 3 is a virus antigen-specific IgG antibody against N protein in cynomolgus monkey serum, and fig. 4 is a virus antigen-specific IgG antibody against RBD protein in cynomolgus monkey serum; in 1-3 groups of animals, the IgG antibody specific to the virus antigen was increased to different degrees at 1 week and 2 weeks compared to before inoculation, and after the second immunization, IgG specific to the virus antigen of the N protein and RBD protein was significantly increased in the serum of 6 cynomolgus monkeys at week 3. In the RBD dimer and trimer groups, virus-specific IgG antibodies against N protein and RBD-RBD protein were significantly elevated. The universal DC vaccine can effectively activate specific antibodies of the cynomolgus monkey against the COVID-19 virus N protein and the RBD protein. The detailed titres are shown in tables 3 and 4.

TABLE 3N-specific IgG antibody titer detection

TABLE 4 RBD-specific IgG antibody titer detection

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Beijing ancient cooking peptide source Biotechnology Ltd

Jiao Shunchang

Fusion gene, fusion protein, recombinant vector, universal DC vaccine of <120> coronavirus and preparation method thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1989

<212> DNA

<213> Artificial Sequence

<400> 1

atgtctgaca acggccctca gaaccagcgg aatgccccaa gaatcacctt cggcggcccc 60

tccgattcta caggctccaa ccagaatgga gagaggtccg gagcacgctc taagcagcgg 120

agaccacagg gcctgcccaa caataccgcc agctggttca ccgccctgac acagcacggc 180

aaggaggacc tgaagtttcc caggggccag ggcgtgccta tcaacaccaa tagctcccct 240

gacgatcaga tcggctacta taggagggca acaaggagaa tccggggagg cgacggcaag 300

atgaaggatc tgtcccccag atggtacttc tactatctgg gaaccggacc tgaggcagga 360

ctgccatatg gcgccaataa ggacggaatc atctgggtgg caaccgaggg cgccctgaac 420

acaccaaagg atcacatcgg cacacgcaat cccgccaaca atgcagcaat cgtgctgcag 480

ctgccacagg gaaccacact gcccaagggc ttttacgcag agggcagcag gggaggctcc 540

caggcctcta gccgctcctc tagccggtcc agaaactcct ctcggaacag caccccaggc 600

agctccaggg gcacaagccc tgcaagaatg gcaggaaacg gaggcgacgc cgccctggcc 660

ctgctgctgc tggatagact gaatcagctg gagtctaaga tgagcggcaa gggacagcag 720

cagcagggac agaccgtgac aaagaagtct gccgccgagg ccagcaagaa gccaaggcag 780

aagcgcaccg ccacaaaggc ctacaacgtg acccaggcct tcggcaggcg cggaccagag 840

cagacacagg gcaattttgg cgaccaggag ctgatcaggc agggcaccga ttataagcac 900

tggcctcaga tcgcacagtt cgcaccaagc gcctccgcct tctttggcat gagcaggatc 960

ggaatggagg tgaccccatc cggcacatgg ctgacctaca caggcgccat caagctggac 1020

gataaggacc ctaacttcaa ggatcaggtc atcctgctga acaagcacat cgatgcctat 1080

aagacctttc cccctacaga gcccaagaag gacaagaaga agaaggccga tgagacccag 1140

gccctgcctc agagacagaa gaagcagcag accgtgacac tgctgccagc agcagacctg 1200

gacgattttt ccaagcagct gcagcagtct atgtctagcg ccgatagcac ccaggccgga 1260

tccatggaag gtagaggttc tctcctcact tgtggtgatg ttgaagaaaa ccctggtcca 1320

agggtgcagc caaccgagtc tatcgtgcgc tttcctaata tcacaaacct gtgcccattt 1380

ggcgaggtgt tcaacgcaac ccgcttcgcc agcgtgtacg cctggaatag gaagcggatc 1440

agcaactgcg tggccgacta tagcgtgctg tacaactccg cctctttcag cacctttaag 1500

tgctatggcg tgtcccccac aaagctgaat gacctgtgct ttaccaacgt ctacgccgat 1560

tctttcgtga tcaggggcga cgaggtgcgc cagatcgccc ccggccagac aggcaagatc 1620

gcagactaca attataagct gccagacgat ttcaccggct gcgtgatcgc ctggaacagc 1680

aacaatctgg attccaaagt gggcggcaac tacaattatc tgtaccggct gtttagaaag 1740

agcaatctga agcccttcga gagggacatc tctacagaaa tctaccaggc cggcagcacc 1800

ccttgcaatg gcgtggaggg ctttaactgt tatttcccac tccagtccta cggcttccag 1860

cccacaaacg gcgtgggcta tcagccttac cgcgtggtgg tgctgagctt tgagctgctg 1920

cacgccccag caacagtgtg cggccccaag aagtccacca atctggtgaa gaacaagtgc 1980

gtgaacttc 1989

<210> 2

<211> 2151

<212> DNA

<213> Artificial Sequence

<400> 2